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目的探讨镁离子对黏液型铜绿假单胞菌早期黏附和生物膜形成过程的影响。方法荧光多功能酶标仪检测各组不同时间点96孔板底部黏附细菌的荧光强度,荧光探针FITC-ConA染细菌胞外多糖(ExtracellularPolymeric Substances,EPS)、荧光显微镜下观察各组多糖差别;SYTO9/PI染生物膜内细菌、激光共聚焦显微镜观察结合BF图像结构分析软件(Image Structor Analyzer,ISA)对各组生物膜结构参数进行定量分析。结果2 d时,空白组和1 mmol/L镁组的黏附细菌的荧光强度分别为1845.67±45.3和2254.78±42.45,t=-9.96,P<0.05;0.1mmol/L的镁浓度下荧光强度也有增加,其余各时间组趋势与2 d组相似;在荧光显微镜下观察可见随着镁浓度增加,EPS增多;激光共聚焦显微镜下可见随着镁浓度增加,生物膜活菌增加、菌落变密集;ISA软件分析结果示:空白组和1 mmol/L镁组的6 d生物膜厚度分别为(25.80±1.16)μm和(34.87±1.59)μm,t=-13.85,P<0.05;区域孔率分别为0.96±0.05和0.90±0.04,t=2.48,P<0.05;平均扩散距离分别为1.54±0.15和1.92±0.16,t=5.23,P<0.05;结构熵分别为3.64±0.57和4.70±1.09,t=-2.6,P<0.05,3 d组生物膜也有相同的趋势。结论镁离子可以增强黏液型铜绿假单胞菌的早期黏附,影响随后生物膜的形成及结构。
Objective To investigate the effect of magnesium ions on the adhesion and biofilm formation of mucoid Pseudomonas aeruginosa at early stage. Methods Fluorescence multifunctional microplate reader was used to detect the fluorescence intensity of the adhered bacteria at the bottom of 96-well plates at different time points. The FITC-ConA stain of extracellular Polymeric Substances (EPS) was observed under fluorescent microscope. SYTO9 / PI staining biofilm bacteria, confocal laser scanning microscopy combined with BF image structure analysis software (Image Structor Analyzer, ISA) quantitative analysis of the biofilm structure parameters of each group. Results At 2 days, the fluorescence intensity of adherent bacteria in blank group and 1 mmol / L magnesium group were 1845.67 ± 45.3 and 2254.78 ± 42.45, respectively, t = -9.96, P <0.05; the fluorescence intensity at 0.1 mmol / L magnesium concentration was also And the trend of the rest time was similar to that of 2 d group. Under the fluorescence microscope, EPS increased with the increase of magnesium concentration. Under confocal laser scanning microscope, with the increase of magnesium concentration, the viable biofilm increased and the colonies became denser. The results of ISA software showed that the 6-day biofilm thickness of blank group and 1 mmol / L magnesium group were (25.80 ± 1.16) μm and (34.87 ± 1.59) μm respectively, t = -13.85, P < (P <0.05). The average diffusion distances were 1.54 ± 0.15 and 1.92 ± 0.16 respectively, t = 5.23, P <0.05. The entropy of structure was 3.64 ± 0.57 and 4.70 ± 1.09 respectively, t = -2.6, P <0.05, 3 d group biofilm also have the same trend. Conclusion Magnesium ions can enhance the early adhesion of mucoid Pseudomonas aeruginosa and affect the subsequent biofilm formation and structure.